Cultured plant cells and plants (tobacco and petunia) were transformed to express green fluorescent protein (GFP) with and without a mitochondrial signal peptide. The localization of GFP in the mitochondria was observed in both cultured tobacco cells and intact transformed tissue which expressed the signal peptide -GFP fusion protein, as determined by colocalization with standard mitochondrial dyes. This technique will be used for microscopic studies of mitochondria localization during meiosis and for studies of the mitochondria signal peptide sequence. The use of two photon excitation microscopy increases the imaging depth, reduces tissue overall damage and enables a more accurate separation of plant autofluorescence from GFP fluorescence since the excitation is far removed from the fluorescence region.